TW202135805A - Pharmaceutical compositon for preventing or treating fibrosis, containing pheophorbide compound as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating fibrosis, containing a pheophorbide compound as an active ingredient. The pharmaceutical composition can effectively inhibit fibrosis of tissues by inhibiting signaling of TGF-β which causes fibrosis and inhibiting activation and expression of collagen and fibronectin, and is significantly superior in the anti-fibrotic activity compared to nintedanib or pirfenidone, which are commercially available therapeutic agents for pulmonary fibrosis, and therefore may be widely used in the prevention or treatment of fibrosis.

Description

Translated fromChinese

含有作為活性成分之去鎂葉綠素酸化合物的用於預防或治療纖維化的藥學組成物Pharmaceutical composition for preventing or treating fibrosis containing pheophytin acid compound as an active ingredient

發明領域Field of invention

本發明係關於一種包含作為活性成分之去鎂葉綠素酸化合物的用於預防或治療纖維化的藥學組成物。The present invention relates to a pharmaceutical composition for preventing or treating fibrosis containing a pheophytin acid compound as an active ingredient.

發明背景Background of the invention

在西方社會中，纖維化疾病佔約45%死因，屬於預防性治療受到限制之嚴重疾病領域，因為其可在疾病取得顯著臨床進展之後被診斷出，且迄今為止無可供使用的直接解決進行性及預先存在之纖維化的有效治療手段。In Western society, fibrotic diseases account for about 45% of deaths and belong to the field of serious diseases where preventive treatment is restricted, because they can be diagnosed after the disease has made significant clinical progress, and there is no direct solution available so far. An effective treatment for sexual and pre-existing fibrosis.

作為異常活化之纖維母細胞的肌纖維母細胞報導為控管纖維化疾病進展之致病細胞。肌纖維母細胞之特徵在於引起由纖維第1,3型膠原蛋白及纖維連接蛋白組成的細胞外基質(ECM)在組織中聚集，且使降解細胞外基質之酶基因失活。Myofibroblasts, which are abnormally activated fibroblasts, are reported as pathogenic cells that control the progression of fibrotic diseases. Myofibroblasts are characterized by causing extracellular matrix (ECM) composed offibrous type 1, 3 collagen and fibronectin to accumulate in tissues and inactivating enzyme genes that degrade the extracellular matrix.

已知生長因子及細胞介素參與肌纖維母細胞之分化及活化，生長因子諸如轉型生長因子-β1(transforming growth factor-beta 1，TGF-β1)、腫瘤壞死因子-α(tumor necrosis factor-alpha，TNF-α)、介白素-1(interleukin-1，IL-1)、IL-6、IL-13、血小板衍生生長因子(platelet-derived growth factor，PDGF)等，由受損的上皮、內皮、骨髓衍生纖維蛋白細胞、免疫細胞及其類似者分泌。It is known that growth factors and cytokines are involved in the differentiation and activation of myofibroblasts. Growth factors such as transforming growth factor-beta 1 (TGF-β1) and tumor necrosis factor-alpha (tumor necrosis factor-alpha, TNF-α), interleukin-1 (IL-1), IL-6, IL-13, platelet-derived growth factor (platelet-derived growth factor, PDGF), etc., are composed of damaged epithelium and endothelium , Bone marrow-derived fibrin cells, immune cells and the like are secreted.

因此，阻斷增加纖維化之生長因子及細胞介素的活化及信號傳導可為治療纖維化之起點，且在本領域中研發靶向TGF-β1、IL-13、結締組織生長因子(connective-tissue growth factor，CTGF)、PDGF、αvβ6整合素、半乳糖凝集素-3、離胺醯氧化酶同源物2(lysyl oxidase homolog 2，LOXL2)、轉麩醯胺酸酶-2、NADPH氧化酶4(NADPH oxidase 4，NOX4)或Jun N端激酶(Jun N-terminal kinase，JNK)抑制因子的藥物用以治療纖維化疾病。Therefore, blocking the activation and signal transduction of growth factors and cytokines that increase fibrosis can be the starting point for the treatment of fibrosis, and the development of targeting TGF-β1, IL-13, connective tissue growth factor (connective- tissue growth factor, CTGF), PDGF, αvβ6 integrin, galectin-3, lysyl oxidase homolog 2 (LOXL2), transglutaminase-2, NADPH oxidase 4 (NADPH oxidase 4, NOX4) or Jun N-terminal kinase (JNK) inhibitor drugs are used to treat fibrotic diseases.

當前，向患有特發性肺纖維化之患者投予的藥物單獨或組合使用類固醇及免疫抑制劑。儘管此等藥物在總計患者之約10-30%中呈現出治療功效，但由於中值存活率小於3年(其無效)及許多副作用，其使用率逐漸下降。Currently, drugs administered to patients with idiopathic pulmonary fibrosis use steroids and immunosuppressants alone or in combination. Although these drugs show therapeutic efficacy in about 10-30% of the total patients, their use rate is gradually decreasing due to a median survival rate of less than 3 years (which is ineffective) and many side effects.

另一方面，在2011準則中，建議吡非尼酮(pirfenidone)(一種抑制TGF-β之啟動子反應的藥物)用於條件式禁止使用。然而，近來其已更新成有條件地可用之藥物，且在動物研究中已展示出減少肌纖維母細胞之擴散。然而，後續臨床測試未報導顯著效果及出現諸如噁心、嘔吐、消化不良及其類似者之副作用。On the other hand, in the 2011 guidelines, pirfenidone (a drug that inhibits the promoter response of TGF-β) is recommended for conditional prohibition. However, it has recently been updated to a conditionally available drug, and it has been shown to reduce the proliferation of myofibroblasts in animal studies. However, subsequent clinical tests did not report significant effects and side effects such as nausea, vomiting, dyspepsia and the like.

另外，尼達尼布(nintedanib)(一種酪胺酸激酶抑制劑)係抑制各種細胞之生長因子受體的藥物，且已知其抑制細胞介素在細胞層面促進纖維化、藉由TGF-β減少膠原蛋白合成及在纖維誘導之動物實驗中緩解肺纖維化之作用。在臨床試驗中，其已展示在廣泛範圍之患者組中延遲肺功能減退之作用，但已知其不能治療肺纖維化。In addition, nintedanib (a tyrosine kinase inhibitor) is a drug that inhibits growth factor receptors in various cells, and it is known to inhibit cytokines to promote fibrosis at the cellular level, and through TGF-β Reduce collagen synthesis and alleviate pulmonary fibrosis in animal experiments induced by fiber. In clinical trials, it has been shown to delay pulmonary hypofunction in a wide range of patient groups, but it is known to be unable to treat pulmonary fibrosis.

因此，吡非尼酮及尼達尼布聚焦於延遲肺功能減退且具有其不充當阻止自身纖維化進展之藥物的限制。因此，本發明者已做出廣泛努力以研發可有效藉由抑制組織纖維化來預防或治療纖維化之藥劑。因此，本發明人已藉由證實去鎂葉綠素酸a抑制TGF-β信號傳導且有效抑制細胞外基質膠原蛋白及纖維連接蛋白之活化來完成本發明，細胞外基質膠原蛋白及纖維連接蛋白為引起纖維化疾病之主要蛋白質。Therefore, pirfenidone and nintedanib focus on delaying lung function decline and have the limitation that they do not act as drugs to prevent the progression of self-fibrosis. Therefore, the inventors have made extensive efforts to develop agents that can effectively prevent or treat fibrosis by inhibiting tissue fibrosis. Therefore, the present inventors have completed the present invention by confirming that pheophytin a inhibits TGF-β signal transduction and effectively inhibits the activation of extracellular matrix collagen and fibronectin. The extracellular matrix collagen and fibronectin are caused by The main protein of fibrotic diseases.

發明概要技術難題Summary of InventionTechnical Difficulties

本發明之一個目的為提供一種用於預防或治療纖維化之藥學組成物，其含有用以藉由抑制組織纖維化而有效預防及治療纖維化的作為活性成分之化合物。An object of the present invention is to provide a pharmaceutical composition for preventing or treating fibrosis, which contains a compound as an active ingredient for effectively preventing and treating fibrosis by inhibiting tissue fibrosis.

本發明之另一目的為提供一種用於預防或改善纖維化之食品組成物，其含有以上作為活性成分之化合物。問題的解決方案Another object of the present invention is to provide a food composition for preventing or improving fibrosis, which contains the above compounds as active ingredients.The solution to the problem

為實現以上目的，本發明提供一種用於預防或治療纖維化之藥學組成物，其含有作為活性成分之去鎂葉綠素酸化合物。In order to achieve the above objectives, the present invention provides a pharmaceutical composition for preventing or treating fibrosis, which contains a pheophytin acid compound as an active ingredient.

本發明亦提供一種用於預防或改善纖維化之食品組成物，其含有作為活性成分之去鎂葉綠素酸化合物。本發明之功效The present invention also provides a food composition for preventing or improving fibrosis, which contains a pheophytin acid compound as an active ingredient.Effect of the present invention

根據本發明的用於預防或治療纖維化之藥學組成物可藉由抑制引起纖維化之TGF-β信號傳導及抑制膠原蛋白及纖維連接蛋白之活化及表現而有效地抑制組織纖維化。另外，由於其展示的抗纖維化活性顯著優於作為市售肺纖維化治療劑的尼達尼布或吡非尼酮，因此其可適用於預防或治療纖維化。The pharmaceutical composition for preventing or treating fibrosis according to the present invention can effectively inhibit tissue fibrosis by inhibiting TGF-β signaling that causes fibrosis and inhibiting the activation and expression of collagen and fibronectin. In addition, since its anti-fibrosis activity is significantly better than nintedanib or pirfenidone, which are commercially available therapeutic agents for pulmonary fibrosis, it can be suitable for preventing or treating fibrosis.

較佳實施例之詳細說明用於預防或治療纖維化之藥學組成物Detailed description of the preferred embodimentPharmaceutical composition for preventing or treating fibrosis

本發明之一個態樣提供一種用於預防或治療纖維化之藥學組成物，其包含作為活性成分之去鎂葉綠素酸化合物。One aspect of the present invention provides a pharmaceutical composition for preventing or treating fibrosis, which contains a pheophytin acid compound as an active ingredient.

在本發明中，術語「去鎂葉綠素酸化合物」具有下式之結構：[式1]

其中R¹、R²及R³各自為選自由以下組成之群的任一者：H、C₁-C₄直鏈或分支鏈烷基、羧基、C₁-C₄烷氧基羰基、C₁-C₄烷氧基羰基C₁-C₄烷基、胺基、胺基-C₁-C₄烷基及側氧基(=O)；以及R⁴係選自由以下組成之群的任一者：H、羥基、側氧基(=O)、C₁-C₄直鏈或分支鏈烷基、C₁-C₄直鏈或分支鏈烷氧基、胺基及胺基-C₁-C₄烷基。In the present invention, the term "pheophytin acid compound" has a structure of the following formula: [Formula 1]

Wherein R¹ , R² and R³ are each selected from the group consisting of H, C₁ -C₄ linear or branched chain alkyl, carboxyl, C₁ -C₄ alkoxycarbonyl, C_1- C₄ alkoxycarbonyl C₁ -C₄ alkyl, amino, amino-C₁ -C₄ alkyl and pendant oxy (=O); and R⁴ is any selected from the group consisting of One: H, hydroxyl, pendant oxy (=O), C₁ -C₄ linear or branched alkyl, C₁ -C₄ linear or branched alkoxy, amine and amino -C₁ -C₄ alkyl.

在本發明中，去鎂葉綠素酸化合物可為去鎂葉綠素酸a，但不限於此。In the present invention, the pheophorbide compound may be pheophorbide a, but is not limited thereto.

在本發明中，術語「去鎂葉綠素酸a」為由C₃₅H₃₆N₄O₅之分子式表示之化合物且具有下式之結構：[式2]

In the present invention, the term "pheophytin a"_{is a compound represented by the molecular formula of C 35} H₃₆ N₄ O₅ and has a structure of the following formula: [Formula 2]

去鎂葉綠素酸化合物，尤其去鎂葉綠素酸a，可抑制TGF-β信號傳導且藉此抑制肌纖維母細胞分化及活化。因此，該化合物可抑制作為肌纖維母細胞之主要標記物的α-SMA之表現，以及作為以纖維化為特徵之細胞外基質蛋白的膠原蛋白及纖維連接蛋白之表現。Pheophytin acid compounds, especially Pheophytin a, can inhibit TGF-β signaling and thereby inhibit myofibroblast differentiation and activation. Therefore, the compound can inhibit the expression of α-SMA, which is the main marker of myofibroblasts, and the expression of collagen and fibronectin, which are extracellular matrix proteins characterized by fibrosis.

在本發明中，術語「TGF-β」係指引起及終止受損組織之修復的關鍵細胞介素，且TGF-β之持續產生致使組織纖維化。特定言之，在慢性肝病的肝組織發現中TGF-β1 mRNA之表現與膠原蛋白I mRNA之表現密切相關。另外，TGF-β1蛋白僅在纖維化取得進展的區域中表現，且不在正常肝組織或非活性區域中表現。因此，已知TGF-β在肝纖維化及肝硬化方面起重要作用。In the present invention, the term "TGF-β" refers to a key cytokine that causes and terminates the repair of damaged tissues, and the continuous production of TGF-β leads to tissue fibrosis. Specifically, the expression of TGF-β1 mRNA in liver tissues of chronic liver disease is closely related to the expression of collagen I mRNA. In addition, TGF-β1 protein is only expressed in areas where fibrosis has progressed, and is not expressed in normal liver tissues or inactive areas. Therefore, it is known that TGF-β plays an important role in liver fibrosis and cirrhosis.

此外，已知TGF-β參與作為纖維化之主要致病細胞的肌纖維母細胞之分化及活化。在纖維化過程之活化中，分子機制之關鍵為藉由TGF-β及Smad依賴性信號傳導活化肌纖維母細胞。TGF-β1與存在於細胞膜中之TGF-β1受體的結合產生TGF-β1之特異性功能的信號傳輸。另外，活化TGF-β1受體誘導Smad2及Smad3蛋白之磷酸化反應，且磷酸化Smad2/Smad3結合至Smad4蛋白以形成複合體。已知Smad複合物易位至細胞核中且引起構成細胞外基質(諸如纖維連接蛋白及膠原蛋白)之蛋白質的轉錄。In addition, it is known that TGF-β participates in the differentiation and activation of myofibroblasts, which are the main pathogenic cells of fibrosis. In the activation of fibrosis, the key to the molecular mechanism is the activation of myofibroblasts through TGF-β and Smad-dependent signal transduction. The binding of TGF-β1 to the TGF-β1 receptor present in the cell membrane produces the signal transmission of the specific function of TGF-β1. In addition, activation of TGF-β1 receptor induces phosphorylation of Smad2 and Smad3 proteins, and phosphorylated Smad2/Smad3 binds to Smad4 protein to form a complex. It is known that the Smad complex translocates into the nucleus and causes the transcription of proteins that make up the extracellular matrix, such as fibronectin and collagen.

因此，去鎂葉綠素酸化合物，尤其去鎂葉綠素酸a，可由抑制TGF-β信號傳導及抑制Smad蛋白磷酸化，從而抑制構成細胞外基質之纖維連接蛋白及膠原蛋白的表現來表徵。Therefore, pheophorbide compounds, especially pheophorbide a, can be characterized by inhibiting TGF-β signaling and inhibiting Smad protein phosphorylation, thereby inhibiting the expression of fibronectin and collagen that constitute the extracellular matrix.

如本文所用，術語「纖維化」意謂過多纖維結締組織形成於器官或組織中。其可區別於作為器官或組織中之正常組分之纖維組織。纖維化可理解為致命疾病，其中藉由纖維母細胞，諸如纖維連接蛋白、膠原蛋白等之細胞外基質積聚過多，從而引起由於器官組織硬化所致的人類組織功能之持續損失。As used herein, the term "fibrosis" means that excessive fibrous connective tissue is formed in an organ or tissue. It can be distinguished from fibrous tissue which is a normal component of organs or tissues. Fibrosis can be understood as a fatal disease, in which fibroblasts, such as fibronectin, collagen, etc., accumulate too much extracellular matrix, which causes the continuous loss of human tissue function due to organ tissue sclerosis.

纖維化可例如出現在選自由以下組成之群的任一或多者中：腎、肝、肺、皮膚、心臟、胰腺、泌尿系統、生殖系統、汗腺、神經、腦、骨髓、肌肉及關節。Fibrosis may occur, for example, in any one or more selected from the group consisting of kidney, liver, lung, skin, heart, pancreas, urinary system, reproductive system, sweat glands, nerves, brain, bone marrow, muscles, and joints.

在本發明中，纖維化可為選自由(但不限於)肝纖維化、肺纖維化、皮膚纖維化、關節纖維化、神經纖維化、胰纖維化、腎纖維化、肌肉纖維化及腹膜纖維化組成之群的任何一或多者。In the present invention, fibrosis may be selected from (but not limited to) liver fibrosis, lung fibrosis, skin fibrosis, joint fibrosis, neurofibrosis, pancreatic fibrosis, kidney fibrosis, muscle fibrosis, and peritoneal fiber Any one or more of the group of chemistry.

更特定言之，纖維化可為選自由(但不限於)以下組成之群的任何一或多者：肺纖維化、特發性肺纖維化、放射誘導之肺損傷或肺纖維化、肺水腫、囊腫纖維化、肝纖維化、心內膜心肌纖維化、心肌梗塞、動脈纖維化、膠質疤痕、腎纖維化、骨髓纖維化、關節纖維化、脂肪纖維化、皮膚纖維化、神經纖維化及肌肉纖維化。More specifically, fibrosis can be any one or more selected from the group consisting of but not limited to: pulmonary fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury or pulmonary fibrosis, pulmonary edema , Cystic fibrosis, liver fibrosis, endocardial fibrosis, myocardial infarction, arterial fibrosis, glial scars, renal fibrosis, bone marrow fibrosis, joint fibrosis, fatty fibrosis, skin fibrosis, neurofibrosis and Muscle fibrosis.

如本文所用，術語「肝纖維化」係指由肝慢性損傷引起的纖維組織之增殖症狀，且可包括但不限於由於選自由以下組成之群的任何一或多者而引起的肝纖維化症狀：慢性肝病、B型肝炎病毒感染、C型肝炎病毒感染、D型肝炎病毒感染、血吸蟲病、酒精性肝病或非酒精性脂肪變性肝炎、代謝疾病、蛋白質缺乏、冠狀動脈疾病、自體免疫性肝炎、囊腫纖維化、α-1抗胰蛋白酶缺乏、原發性膽汁性肝硬化、藥物反應及毒素。As used herein, the term "liver fibrosis" refers to the symptoms of proliferation of fibrous tissue caused by chronic liver injury, and may include, but is not limited to, symptoms of liver fibrosis caused by any one or more selected from the following groups : Chronic liver disease, hepatitis B virus infection, hepatitis C virus infection, hepatitis D virus infection, schistosomiasis, alcoholic liver disease or non-alcoholic steatosis hepatitis, metabolic disease, protein deficiency, coronary artery disease, autoimmunity Hepatitis, cystic fibrosis, alpha-1 antitrypsin deficiency, primary biliary cirrhosis, drug reactions and toxins.

肝纖維化為肝硬化前病，且藉由各種細胞介素及生長因子之作用而開始，該作用由引起慢性肝病之嚴重肝臟損傷所致。一般而言，肝纖維化由可逆、薄原纖維組成，且若無節結形成且肝損傷原因係暫時的，則增加之細胞外基質(ECM)藉由細胞凋亡及基質金屬蛋白酶(MMP)降解以允許正常恢復。然而，肝纖維化過程之重複延續使得形成厚原纖維且進展至節點肝硬化。另外，肝硬化經由肝纖維化過程誘導，其中肝細胞受各種發炎誘導因子破壞且包括膠原蛋白之異常細胞外基質蛋白積聚。因此，重要的是控制細胞外基質之積聚以調節肝硬化表現。在破壞肝細胞之情況下之發炎反應活化休眠階段中之肝星形細胞，以分泌細胞外基質及各種細胞介素及趨化激素，其中TGF-β1充當強效生長抑制劑。藉由結合至潛伏TGF-β1結合蛋白，TGF-β1(一種25 kD物質)以無活性潛伏形式分泌，且以結合至細胞外基質(諸如第1,4型膠原蛋白、層黏連蛋白及核心蛋白聚糖，其藉由各種刺激活化)之形式存在。TGF-β1藉由減少膠原蛋白酶產生或增加膠原蛋白酶抑制劑產生來調節膠原蛋白表現，增加巨噬細胞中TNF-α、IL-1、PDGF等之產生且在纖維化過程中起重要作用。目前已知TGF-β1僅在纖維化發展之區域中，而不在正常肝組織中或在非活性區域中表現，且在肝纖維化方面起重要作用。Liver fibrosis is a pre-cirrhotic disease and is initiated by the action of various cytokines and growth factors, which are caused by severe liver damage that causes chronic liver disease. Generally speaking, liver fibrosis is composed of reversible, thin fibrils, and if no nodules are formed and the cause of liver damage is temporary, the increased extracellular matrix (ECM) is caused by apoptosis and matrix metalloproteinases (MMP). Degraded to allow normal recovery. However, the repeated continuation of the liver fibrosis process results in the formation of thick fibrils and progression to nodal cirrhosis. In addition, liver cirrhosis is induced through the process of liver fibrosis, in which liver cells are destroyed by various inflammation-inducing factors and abnormal extracellular matrix proteins including collagen accumulate. Therefore, it is important to control the accumulation of extracellular matrix to regulate the performance of liver cirrhosis. The inflammatory response in the case of destruction of hepatocytes activates hepatic stellate cells in the dormant phase to secrete extracellular matrix and various cytokines and chemotactic hormones, among which TGF-β1 acts as a potent growth inhibitor. By binding to the latent TGF-β1 binding protein, TGF-β1 (a 25 kD substance) is secreted in an inactive latent form and binds to extracellular matrix (such ascollagen types 1, 4, laminin, and core Proteoglycans exist in the form of activation by various stimuli. TGF-β1 regulates collagen expression by reducing the production of collagenase or increasing the production of collagenase inhibitors, increases the production of TNF-α, IL-1, PDGF, etc. in macrophages, and plays an important role in the process of fibrosis. It is currently known that TGF-β1 is only present in areas where fibrosis develops, but not in normal liver tissues or in inactive areas, and it plays an important role in liver fibrosis.

另一方面，非酒精性脂肪肝，無論飲酒與否，可由肥胖症、糖尿病、高脂血症、藥物及其類似者引起，且涵蓋各種疾病，包括不伴隨發炎反應之單純性脂肪肝(脂肪變性)及展示肝細胞發炎、晚期纖維化及肝硬化之非酒精性脂肪變性肝炎(NASH)，其視疾病進展而定。據報導，隨著現代社會中歸因於高脂及高卡路里飲食之成年疾病的增加，發達國家中20-30%之成年群體出現非酒精性脂肪肝病(non-alcoholic fatty liver disease，NAFLD)，其中2-3%發展成非酒精性脂肪變性肝炎(non-alcoholic steatohepatitis，NASH)患者，該等非酒精性脂肪變性肝炎患者尤其呈現在組織學上伴隨有纖維化及發炎之脂肪變性肝炎發現且具有罹患肝硬化、肝衰竭及肝癌之高風險。On the other hand, non-alcoholic fatty liver, whether drinking alcohol or not, can be caused by obesity, diabetes, hyperlipidemia, drugs and the like, and covers various diseases, including simple fatty liver without inflammation (fatty Degeneration) and non-alcoholic steatohepatitis (NASH) showing liver cell inflammation, advanced fibrosis, and cirrhosis, depending on the progression of the disease. According to reports, with the increase in adult diseases attributed to high-fat and high-calorie diets in modern society, 20-30% of adult populations in developed countries suffer from non-alcoholic fatty liver disease (NAFLD). Among them, 2-3% develop non-alcoholic steatohepatitis (NASH) patients. These non-alcoholic steatohepatitis patients especially show histological findings of steatohepatitis accompanied by fibrosis and inflammation. There is a high risk of cirrhosis, liver failure and liver cancer.

如本文所用，術語「肺纖維化」係指歸因於肺中過多纖維結締組織之形成或發展而出現疤痕化(纖維)組織(纖維化)。具體言之，肺纖維化為引起肺泡及肺間質組織腫脹及疤痕形成的慢性疾病。此類疤痕化組織替代健康組織且引起發炎，且慢性發炎可視為纖維化跡象。此類對肺組織之損傷可致使肺僵硬且可使得個體難以維持自發呼吸。As used herein, the term "pulmonary fibrosis" refers to the appearance of scarred (fibrous) tissue (fibrosis) due to the formation or development of excessive fibrous connective tissue in the lung. Specifically, pulmonary fibrosis is a chronic disease that causes swelling and scar formation of alveoli and interstitial tissue. Such scarred tissue replaces healthy tissue and causes inflammation, and chronic inflammation can be regarded as a sign of fibrosis. Such damage to lung tissue can cause lung stiffness and can make it difficult for individuals to maintain spontaneous breathing.

特定言之，肺纖維化可包括但不限於選自由以下組成之群的任何一或多者：特發性肺纖維化；放射誘導之肺損傷；非特異性間質性肺炎；急性間質性肺炎；隱源性機化性肺炎；呼吸性細支氣管炎相關間質性肺病；落屑性間質性肺炎；淋巴間質性肺炎；間質性肺纖維化；及由彌漫性肺纖維化、肺水腫、囊腫性纖維化及代謝疾病引起的肺纖維化。Specifically, pulmonary fibrosis may include, but is not limited to, any one or more selected from the group consisting of: idiopathic pulmonary fibrosis; radiation-induced lung injury; nonspecific interstitial pneumonia; acute interstitial Pneumonia; cryptogenic organizing pneumonia; respiratory bronchiolitis-related interstitial lung disease; squamous interstitial pneumonia; lymphoid interstitial pneumonia; interstitial pulmonary fibrosis; and diffuse pulmonary fibrosis, lung Pulmonary fibrosis caused by edema, cystic fibrosis and metabolic diseases.

如本文所用，術語「皮膚纖維化」係過多皮膚疤痕形成，且為病理性傷口癒合反應之結果。存在廣泛範圍之纖維化皮膚病：硬皮病、腎源性纖維化皮膚病、混合結締組織疾病、硬化性黏液水腫(scleromyxedema)、硬腫病及嗜酸性筋膜炎。暴露於化學品或物理藥劑(機械創傷、燒傷傷口)亦為纖維化皮膚病之潛在原因。皮膚纖維化可由免疫、自體免疫及發炎機制驅動。膠原蛋白產生與纖維母細胞降解之平衡在皮膚纖維化之病理生理學方面起關鍵作用。某些細胞介素，諸如轉型生長因子-b(TGF-β)及介白素-4(IL-4)促進傷口癒合及纖維化。正常皮膚中纖維母細胞為靜息的。其合成受控量之結締組織蛋白且具有低增殖活性。皮膚損傷後，此等細胞變得活化，亦即其表現α-平滑肌肌動蛋白(α-SMA)且合成大量結締組織蛋白。活化細胞通常稱為肌纖維母細胞。此處，「皮膚纖維化」亦可包括「硬皮病」。As used herein, the term "skin fibrosis" refers to excessive skin scarring and is the result of pathological wound healing reactions. There are a wide range of fibrotic skin diseases: scleroderma, nephrogenic fibrotic skin diseases, mixed connective tissue diseases, scleromyxedema, scleromyxedema, and eosinophilic fasciitis. Exposure to chemicals or physical agents (mechanical trauma, burn wounds) is also a potential cause of fibrotic skin diseases. Skin fibrosis can be driven by immune, autoimmune and inflammation mechanisms. The balance between collagen production and fibroblast degradation plays a key role in the pathophysiology of skin fibrosis. Certain cytokines, such as transforming growth factor-b (TGF-β) and interleukin-4 (IL-4), promote wound healing and fibrosis. Fibroblasts in normal skin are resting. It synthesizes a controlled amount of connective tissue protein and has low proliferative activity. After skin injury, these cells become activated, that is, they express α-smooth muscle actin (α-SMA) and synthesize a large amount of connective tissue protein. Activated cells are often called myofibroblasts. Here, "skin fibrosis" may also include "scleroderma".

如本文所用，術語「心臟纖維化」係指歸因於心臟細胞之間基質蛋白質的過度沈積而使心臟硬化的現象。心臟纖維化主要存在於心肌梗塞患者之心臟中且為心臟功能減退之主要原因。心臟纖維化可包括但不限於選自由以下組成之群之任一或多者：心內膜纖維化、心房纖維化、心臟衰竭、心肌梗塞，以及由代謝疾病引起之心臟纖維化。另外，因為心臟纖維化係心臟衰竭及心肌梗塞之主要病因，所以術語「心臟纖維化」可解釋為涵蓋由心臟纖維化引起之心臟衰竭及/或心肌梗塞。As used herein, the term "cardiac fibrosis" refers to a phenomenon in which the heart hardens due to the excessive deposition of matrix proteins between heart cells. Cardiac fibrosis mainly exists in the heart of patients with myocardial infarction and is the main cause of cardiac hypofunction. Cardiac fibrosis may include, but is not limited to, any one or more selected from the group consisting of endocardial fibrosis, atrial fibrosis, heart failure, myocardial infarction, and cardiac fibrosis caused by metabolic diseases. In addition, because cardiac fibrosis is the main cause of heart failure and myocardial infarction, the term "cardiac fibrosis" can be interpreted as covering heart failure and/or myocardial infarction caused by cardiac fibrosis.

在本發明中，藥學組成物可進一步含有藥學上可接受之載劑。In the present invention, the pharmaceutical composition may further contain a pharmaceutically acceptable carrier.

本發明之藥學組成物中所包含之藥學上可接受之載劑為習知用於製備中之彼等載劑，且包括但不限於乳糖、右旋糖、蔗糖、山梨糖醇、甘露糖醇、澱粉、阿拉伯樹膠、磷酸鈣、海藻酸鹽、明膠、矽酸鈣、微晶纖維素、聚乙烯吡咯啶酮、纖維素、水、糖漿、甲基纖維素、甲基羥基苯甲酸酯、丙基羥基苯甲酸酯、滑石、硬脂酸鎂、礦物油、生理食鹽水、磷酸鹽緩衝鹽水(phosphate buffered saline，PBS)或介質及其類似物，且除以上組分之外，可進一步包括潤滑劑、潤濕劑、甜味劑、調味劑、乳化劑、懸浮劑、防腐劑等。The pharmaceutically acceptable carriers contained in the pharmaceutical composition of the present invention are those conventionally used in preparation, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, and mannitol , Starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, Propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, physiological saline, phosphate buffered saline (PBS) or medium and the like, and in addition to the above components, further Including lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

本發明之藥學組成物可藉由用藥學上可接受之載劑及/或賦形劑調配或藉由根據一般熟習此項技術者易於實踐之方法併入至大容量容器中來製備成單位劑型，且可進一步包含分散劑或穩定劑。The pharmaceutical composition of the present invention can be prepared into a unit dosage form by formulating with a pharmaceutically acceptable carrier and/or excipient or by incorporating it into a large-capacity container according to a method easily practiced by those skilled in the art. , And may further include a dispersant or stabilizer.

藥學組成物之調配物可視使用方法而變化，但可製備為石膏、顆粒、粉劑、糖漿、溶液、流浸膏劑、乳液、懸浮液、輸注液、片劑、注射劑、膠囊及丸劑。藥學組成物之調配物亦包括軟膏、糊劑、乳膏、乳劑、凝膠、粉劑、溶液、噴霧劑、吸入劑或貼片，其為用於局部或經皮投予之調配物。The formulation of the pharmaceutical composition can vary depending on the method of use, but can be prepared as plaster, granules, powder, syrup, solution, liquid extract, emulsion, suspension, infusion, tablet, injection, capsule, and pill. The formulations of pharmaceutical compositions also include ointments, pastes, creams, emulsions, gels, powders, solutions, sprays, inhalants or patches, which are formulations for topical or transdermal administration.

藥學組成物可與藥學上可接受之載劑及(必要時)防腐劑、緩衝劑等無菌混合。根據本發明之軟膏、糊劑、乳膏及凝膠調配物可作為賦形劑另外含有動物及植物脂肪、油、蠟、石蠟、澱粉、黃蓍膠、纖維素衍生物、聚乙二醇、聚矽氧、膨潤土、矽酸、滑石、氧化鋅或其混合物。The pharmaceutical composition can be aseptically mixed with pharmaceutically acceptable carriers and (if necessary) preservatives, buffers, etc. The ointments, pastes, creams and gel formulations according to the present invention can be used as excipients and additionally contain animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, Polysiloxane, bentonite, silicic acid, talc, zinc oxide or mixtures thereof.

本發明之藥學組成物當含有有效量之去鎂葉綠素酸a時可提供纖維化之所需預防性、改善性或治療性作用。如本文所用，術語「有效量」係指呈現與陰性對照相比更多的反應的量，較佳為足以預防、改善或治療纖維化的量。在一個實施例中，本發明之藥學組成物可含有0.01%至99.9%之量的去鎂葉綠素酸化合物(去鎂葉綠素酸a)，且可由藥學上可接受之載劑充占餘量。本發明之藥學組成物中所包含之化合物將視組成物商業化之形式及其類似形式而變化。When the pharmaceutical composition of the present invention contains an effective amount of pheophytin a, it can provide the necessary preventive, ameliorating or therapeutic effects of fibrosis. As used herein, the term "effective amount" refers to an amount that exhibits a greater response than the negative control, preferably an amount sufficient to prevent, ameliorate, or treat fibrosis. In one embodiment, the pharmaceutical composition of the present invention may contain 0.01% to 99.9% of the pheophorbide compound (pheophorbide a), and a pharmaceutically acceptable carrier can make up the balance. The compounds contained in the pharmaceutical composition of the present invention will vary depending on the commercialized form of the composition and similar forms.

本發明之藥學組成物之總有效量可以單次劑量或藉由在一段較長時間內投予多次劑量的分開治療方案向患者投予。視疾病程度而定，本發明之藥學組成物可改變活性成分之含量。舉例而言，其可以一至數次分次劑量投予，使得其基於去鎂葉綠素酸a以每公斤體重每天0.001 μg至100 mg，更佳0.01 μg至10 mg之量投予。然而，因為去鎂葉綠素酸a之劑量係藉由考慮各種因素而確定，該等因素諸如年齡、體重、健康狀況、性別、疾病嚴重程度、患者飲食及排泄率以及藥學組成物投予途徑及治療頻率。考慮上述內容，本領域中一般熟習此項技術者將能夠根據預防、治療或改善纖維化之特定用途確定去鎂葉綠素酸a之適當有效劑量。根據本發明之藥學組成物在其調配物、投予途徑及投予方法中不受特別限制，只要實現本發明之作用即可。用於預防或改善纖維化之食品組成物The total effective amount of the pharmaceutical composition of the present invention can be administered to the patient in a single dose or by a separate treatment plan in which multiple doses are administered over a long period of time. Depending on the degree of the disease, the pharmaceutical composition of the present invention can change the content of the active ingredient. For example, it can be administered in one to several divided doses such that it is administered in an amount of 0.001 μg to 100 mg per kilogram of body weight per day based on pheophytin a, and more preferably 0.01 μg to 10 mg. However, because the dosage of pheophytin a is determined by considering various factors, such as age, weight, health status, gender, severity of disease, patient’s diet and excretion rate, and the route of administration and treatment of the pharmaceutical composition frequency. Considering the above content, those skilled in the art will be able to determine the appropriate and effective dose of pheophytin a according to the specific application for preventing, treating or improving fibrosis. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, and administration method, as long as the effect of the present invention is achieved.Food composition for preventing or improving fibrosis

根據本發明之另一態樣，提供一種用於預防或改善纖維化之食品組成物，其含有作為活性成分之去鎂葉綠素酸化合物。According to another aspect of the present invention, there is provided a food composition for preventing or improving fibrosis, which contains a pheophytin acid compound as an active ingredient.

在本發明中，去鎂葉綠素酸化合物可為(但不限於)去鎂葉綠素酸a。In the present invention, the pheophorbide compound may be (but not limited to) pheophorbide a.

在本發明中，術語「食品」意謂含有一或多種養分的天然產品或加工產品，且較佳意謂在經由某些加工步驟處在可直接地食用的狀態下的產品，且該術語包括如一般含義的所有食品、食品添加劑、保健功能性食品、飲料、飲料添加劑及其類似物。In the present invention, the term "food" means a natural product or processed product containing one or more nutrients, and preferably means a product in a state that can be directly eaten through certain processing steps, and the term includes Such as all foods, food additives, health-care functional foods, beverages, beverage additives and the like in the general meaning.

當本發明之組成物製備成食品組成物時，其不僅含有作為活性成分之黃漆木提取物，而且含有通常在食品製備中添加之組分，例如蛋白質、碳水化合物、脂肪、營養物、調味料及調味劑。預防及治療纖維化之方法When the composition of the present invention is prepared into a food composition, it not only contains the yellow lacquer wood extract as an active ingredient, but also contains components that are usually added in food preparation, such as protein, carbohydrate, fat, nutrients, and flavoring. Ingredients and flavoring agents.Methods of preventing and treating fibrosis

本發明之另一態樣提供預防及治療纖維化之方法，其包含向個體投予去鎂葉綠素酸化合物。Another aspect of the present invention provides a method for preventing and treating fibrosis, which comprises administering a pheophytin acid compound to an individual.

個體可為患有纖維化之個體。個體亦可為哺乳動物，較佳人類。在此情況下，去鎂葉綠素酸化合物如上文所述，且較佳地去鎂葉綠素酸化合物可為去鎂葉綠素酸a。另外，化合物之途徑、劑量及投予頻率可視患者之病狀及存在或不存在副作用而變化，化合物可以各種方法及量向個體投予，且最佳投予方法、劑量及投予頻率可由一般熟習此項技術者在適當範圍內選擇。纖維化之類型亦如上文所描述。去鎂葉綠素酸化合物用於治療纖維化之用途The individual may be an individual suffering from fibrosis. The individual may also be a mammal, preferably a human. In this case, the pheophorbide compound is as described above, and preferably the pheophorbide compound may be pheophorbide a. In addition, the route, dosage, and frequency of administration of the compound may vary depending on the patient’s condition and the presence or absence of side effects. The compound can be administered to the individual in various methods and amounts, and the optimal administration method, dosage, and frequency of administration can be determined by general Those who are familiar with this technology should choose within an appropriate range. The type of fibrosis is also as described above.Use of pheophytin acid compound for treating fibrosis

本發明之另一態樣提供去鎂葉綠素酸化合物用於治療纖維化之用途。Another aspect of the present invention provides the use of a pheophytin acid compound for the treatment of fibrosis.

在此情況下，去鎂葉綠素酸化合物如上文所述，且較佳地去鎂葉綠素酸化合物可為去鎂葉綠素酸a。纖維化之類型亦如上文所描述。實施方式In this case, the pheophorbide compound is as described above, and preferably the pheophorbide compound may be pheophorbide a. The type of fibrosis is also as described above.Implementation

在下文中，將經由實例更詳細地描述本發明。然而，對於一般熟習此項技術者將顯而易見的是，以下實例僅用於說明本發明，且本發明的範疇不限於此。製備實例1.黃漆木提取物之製備Hereinafter, the present invention will be described in more detail through examples. However, it will be obvious to those who are generally familiar with the art that the following examples are only used to illustrate the present invention, and the scope of the present invention is not limited thereto.Preparation example 1. Preparation of yellow lacquer wood extract

黃漆木之葉、莖、根等經洗滌且使用研磨機研磨，且隨後將乙醇或甲醇添加至約8 g研磨產物中達到20 w/v%之濃度。將其浸沒且攪拌至少4小時，且隨後過濾以分離初級液體組分。將所分離之固體再次浸沒且在乙醇或甲醇中攪拌至少4小時，且隨後過濾，得到次級液體組分。混合所得初級液體組分及次級液體組分，在減壓下濃縮混合物，且使殘餘物冷凍乾燥，得到黃漆木提取物。製備實例2.自黃漆木提取物分離去鎂葉綠素酸aThe leaves, stems, roots, etc. of the yellow lacquered wood were washed and ground using a grinder, and then ethanol or methanol was added to about 8 g of the ground product to reach a concentration of 20 w/v%. It was submerged and stirred for at least 4 hours, and then filtered to separate the primary liquid component. The separated solid was submerged again and stirred in ethanol or methanol for at least 4 hours, and then filtered to obtain a secondary liquid component. The obtained primary liquid component and the secondary liquid component are mixed, the mixture is concentrated under reduced pressure, and the residue is freeze-dried to obtain a yellow lacquer wood extract.Preparation Example 2. Separation of Pheophyllophyllic Acid a from Yellow Lacquer Extract

將製備實例1中獲得之黃漆木提取物與石油醚、己烷、氯仿、二氯甲烷及乙酸乙酯按如所述次序分級分離以獲得級分。高度活性氯仿級分經受矽膠管柱上石油醚、乙基醚、甲醇及水之溶離條件，獲得總共6個細分級分(F-1至F-6)。其中，F-2級分經受矽膠管柱上石油醚、乙基醚及甲醇之溶離條件，獲得總共10個細分級分(F-1a至F-10a)。The lacquer wood extract obtained in Preparation Example 1 was fractionated with petroleum ether, hexane, chloroform, dichloromethane and ethyl acetate in the order described to obtain fractions. The highly active chloroform fraction was subjected to the dissolution conditions of petroleum ether, ethyl ether, methanol and water on the silica gel column to obtain a total of 6 subdivided fractions (F-1 to F-6). Among them, the F-2 fraction was subjected to the dissolution conditions of petroleum ether, ethyl ether and methanol on the silica gel column to obtain a total of 10 subdivided fractions (F-1a to F-10a).

其中，藉由使F-4a細分級分經受在矽膠管柱上二氯甲烷:甲醇=95:05(v/v)之溶離條件而獲得總共三個細分級分(F-1b至F-3b)。其中，藉由NMR分析在F-3b細分級分中證實去鎂葉綠素酸a，如下表1中所示(圖1)。[表1]位置¹H化學位移A9.42β9.56δ8.602a8.002b6.25106.2784.5074.0510b3.884a3.705a3.651a3.333a3.207a,7b2.30-2.508a1.804b1.65實驗實例1.去鎂葉綠素酸a細胞毒性之實驗Among them, by subjecting the F-4a subdivision fraction to the dissolution condition of methylene chloride:methanol=95:05 (v/v) on a silica gel column, a total of three subdivision fractions (F-1b to F-3b) ). Among them, pheophytin acid a was confirmed in the F-3b sub-fraction by NMR analysis, as shown in Table 1 below (Figure 1). [Table 1]Location¹Hchemical shift A 9.42 β 9.56 δ 8.60 2a 8.00 2b 6.25 10 6.27 8 4.50 7 4.05 10b 3.88 4a 3.70 5a 3.65 1a 3.33 3a 3.20 7a, 7b 2.30-2.50 8a 1.80 4b 1.65 Experimental example 1. Cytotoxicity experiment of pheophytin acid a

為證實製備實例2中所獲得之去鎂葉綠素酸a的細胞毒性，使用作為人類肺衍生纖維母細胞之CCD8-Lu細胞及LL-29細胞。將CCD8-Lu細胞及LL-29細胞以2×10³個細胞/孔接種至96孔培養盤中且在37℃下在濕潤CO₂培育箱中培養24小時情況下穩定。經培養細胞用製備實例2中所獲得之不同濃度之去鎂葉綠素酸a處理，且72小時後，用WST-1試劑處理。在1小時之後，使用微量培養盤讀取器(EZ Read 400，biochrom，UK)量測細胞存活率，以測定450 nm處之吸光度，且評估毒性。To confirm the cytotoxicity of pheophytin a obtained in Preparation Example 2, CCD8-Lu cells and LL-29 cells, which are human lung-derived fibroblasts, were used. CCD8-Lu cells and LL-29 cells were seeded into a 96-well culture dish^{at 2×10 3} cells/well and stable under the condition of culturing_{in a humidified CO 2 incubator at 37° C. for 24 hours.} The cultured cells were treated with the different concentrations of pheophytin a obtained in Preparation Example 2, and after 72 hours, they were treated with WST-1 reagent. After 1 hour, a microplate reader (EZ Read 400, biochrom, UK) was used to measure the cell viability to measure the absorbance at 450 nm and evaluate the toxicity.

藉由表現經樣品處理之組相對於對照組之吸光度之百分比，細胞存活率作為實驗結果展示於圖3中。如圖3中所示，證實去鎂葉綠素酸a直至2.5 μM之濃度為止具有很小細胞毒性，且呈現在5 μM之濃度下約25%及在10 μM之濃度下約75%之細胞毒性。實驗實例2.證實去鎂葉綠素酸a對TGF-β信號傳導途徑之抑制效果By expressing the percentage of absorbance of the sample-treated group relative to the control group, the cell survival rate is shown in Figure 3 as the experimental result. As shown in FIG. 3, it was confirmed that pheophytin a has little cytotoxicity up to a concentration of 2.5 μM, and exhibits cytotoxicity of about 25% at a concentration of 5 μM and about 75% at a concentration of 10 μM.Experimental example 2. Confirmation of the inhibitory effect of pheophytin a on TGF-β signaling pathway

製備載體，其中藉由重複八次Smad可結合之核苷酸序列(5'-GGTGTCTAGACATAGTCTAGAGACA-3' SEQ ID NO: 1)來獲得的基因，與編碼螢光素酶的作為報導基因之基因連接。將上述載體與其中RSV啟動子基因連接至編碼β-半乳糖苷酶之基因的載體一起轉染至Balb/c3T3細胞中。6小時之後，陰性對照組不用處理，陽性對照組用5 ng/ml濃度之TGF-β處理，且實驗組用製備實例2中所獲得之0.06 μM、0.12 μM、0.25 μM、0.5 μM、1 μM及5 μM之各濃度的去鎂葉綠素酸a以及5 ng/ml之TGF-β處理。A vector was prepared in which the gene obtained by repeating the Smad-binding nucleotide sequence (5'-GGTGTCTAGACATAGTCTAGAGACA-3' SEQ ID NO: 1) eight times was linked to a gene encoding luciferase as a reporter gene. The above vector was transfected into Balb/c3T3 cells together with a vector in which the RSV promoter gene was linked to the gene encoding β-galactosidase. After 6 hours, the negative control group was left untreated, the positive control group was treated with TGF-β at a concentration of 5 ng/ml, and the experimental group was treated with the 0.06 μM, 0.12 μM, 0.25 μM, 0.5 μM, and 1 μM obtained in Preparation Example 2. And 5 μM pheophytin a at each concentration and 5 ng/ml TGF-β treatment.

細胞接著在5% CO₂培育箱中在37℃下培養20小時，且接著進行螢光素酶分析。因此，如圖4中所示，證實蛋白質之活性藉由Balb/c3T3細胞中之TGF-β增加，但藉由TGF-β增加之Smad蛋白的活性藉由去鎂葉綠素酸a以濃度依賴性方式降低。因此，發現經由TGF-β信號傳導活化之Smad的磷酸化藉由去鎂葉綠素酸a抑制。實驗實例3.對去鎂葉綠素酸a化合物之抗纖維化活性的分析實驗實例3.1.使用西方墨點法分析進行對抗纖維化活性之分析The cells were then cultured in a 5% CO₂ incubator at 37°C for 20 hours, and then subjected to luciferase analysis. Therefore, as shown in Figure 4, it was confirmed that the activity of the protein was increased by TGF-β in Balb/c3T3 cells, but the activity of the Smad protein increased by TGF-β was increased by pheophytin a in a concentration-dependent manner reduce. Therefore, it was found that the phosphorylation of Smad activated by TGF-β signaling is inhibited by pheophytin a. Experimental example 3. Analysis of the anti-fibrotic activity of pheophytin acid a compound Experimental example 3.1. Analysis of anti-fibrotic activity using Western blot analysis

使用西方墨點法分析進行基於細胞之分析實驗，以分析去鎂葉綠素酸a之抗纖維化活性。Western blot analysis was used to perform cell-based analysis experiments to analyze the anti-fibrotic activity of pheophytin a.

在含有10% FBS之DMEM培養基中培養作為人類肺衍生纖維母細胞的CCD8-Lu細胞。隨後，將約2×10⁵個細胞接種至6孔培養盤中且培養24小時，且在移除FBS之DMEM培養基中經受缺乏條件約24小時。CCD8-Lu cells, which are human lung-derived fibroblasts, were cultured in DMEM medium containing 10% FBS. Subsequently, about 2×10⁵ cells were seeded into a 6-well culture dish and cultured for 24 hours, and subjected to the lack of conditions for about 24 hours in DMEM medium with FBS removed.

24小時後，在用濃度為5 ng/ml之TGF-β處理之後，誘導細胞纖維化持續6小時。6小時後，細胞用製備實例2中所獲得之去鎂葉綠素酸a處理且進一步培養18小時(圖2)。接著，陰性對照組不用處理，陽性對照組僅用5 ng/ml濃度之TGF-β處理，且實驗組用5 mg/mL濃度之TGF-β處理，並在6小時後，用濃度為0.06 μM、0.12 μM、0.25 μM、0.5 μM、2.5 μM及5 μM之去鎂葉綠素酸a處理。After 24 hours, after treatment with TGF-β at a concentration of 5 ng/ml, cell fibrosis was induced for 6 hours. After 6 hours, the cells were treated with pheophytin a obtained in Preparation Example 2 and further cultured for 18 hours (Figure 2). Then, the negative control group was not treated, the positive control group was only treated with TGF-β at a concentration of 5 ng/ml, and the experimental group was treated with TGF-β at a concentration of 5 mg/mL, and after 6 hours, with a concentration of 0.06 μM , 0.12 μM, 0.25 μM, 0.5 μM, 2.5 μM and 5 μM pheophytin a treatment.

18小時後，移除培養基且藉由添加PBS緩衝溶液洗滌細胞二次，且直接向其中添加含有蛋白酶抑制劑之100 μl RIPA緩衝溶液(150 mM NaCl、1% NP-40、0.5%去氧膽酸、0.1% SDS、50 mM Tris pH 7.5)。10分鐘後，使用刮刀回收細胞且轉移至微管中且以約12,000×g離心10分鐘。After 18 hours, remove the medium and wash the cells twice by adding PBS buffer solution, and directly add 100 μl RIPA buffer solution containing protease inhibitors (150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid) to it. Acid, 0.1% SDS, 50 mM Tris pH 7.5). After 10 minutes, the cells were recovered using a spatula and transferred to a microtube and centrifuged at approximately 12,000×g for 10 minutes.

獲得經分離之上清液且轉移至新的微管中，且接著使用二喹啉甲酸分析(bicinchoninic acid assay，BCA分析)蛋白質定量套組來定量該蛋白質。對具有相同量蛋白質之各樣品進行電泳，且隨後進行西方墨點法。抗膠原蛋白1A或抗纖維連接蛋白、抗α-平滑肌肌動蛋白抗體用作一級抗體，且抗小鼠IgG HRP或抗兔IgG HRP用作二級抗體。使用用於校正的抗β-肌動蛋白抗體來校正蛋白質表現之差異。The separated supernatant was obtained and transferred to a new microtube, and then the protein was quantified using the bicinchoninic acid assay (BCA analysis) protein quantification kit. Electrophoresis was performed on each sample with the same amount of protein, and then the Western blot method was performed. Anti-collagen 1A or anti-fibronectin, anti-α-smooth muscle actin antibodies were used as primary antibodies, and anti-mouse IgG HRP or anti-rabbit IgG HRP were used as secondary antibodies. Use the anti-β-actin antibody for correction to correct for differences in protein performance.

根據圖5中所示之實驗結果，發現在用TGF-β處理作為人類肺衍生纖維母細胞的CCD8-Lu細胞時，膠原蛋白1A、纖維連接蛋白及平滑肌肌動蛋白之表現增加，且證實由TGF-β誘導之膠原蛋白1A、纖維連接蛋白及平滑肌肌動蛋白蛋白的表現藉由用去鎂葉綠素酸a治療而抑制。因此，去鎂葉綠素酸a抑制以纖維化為特徵之蛋白質的表現，從而證實遏制纖維化進展之可能性。實驗實例3.2.使用免疫細胞化學染色分析進行對抗纖維化活性之分析According to the experimental results shown in Figure 5, it was found that when CCD8-Lu cells, which are human lung-derived fibroblasts, were treated with TGF-β, the expression of collagen 1A, fibronectin, and smooth muscle actin increased, and it was confirmed that The expression of collagen 1A, fibronectin and smooth muscle actin protein induced by TGF-β was inhibited by treatment with pheophytin a. Therefore, pheophytin a inhibits the expression of proteins characterized by fibrosis, thus confirming the possibility of curbing the progress of fibrosis.Experimental example 3.2. Analysis of anti-fibrosis activity using immunocytochemical staining analysis

使用免疫細胞化學染色方法進行基於細胞之分析實驗，以分析製備實例2中獲得之去鎂葉綠素酸a的抗纖維化活性。The immunocytochemical staining method was used to perform cell-based analysis experiments to analyze the anti-fibrotic activity of pheophytin a obtained in Preparation Example 2.

在含有10% FBS之DMEM培養基中培養作為人類肺衍生纖維母細胞的CCD8-Lu細胞。隨後將約2×10⁴個細胞接種至24孔培養盤中且培養24小時，且在移除FBS之DMEM培養基中經受缺乏條件約24小時。CCD8-Lu cells, which are human lung-derived fibroblasts, were cultured in DMEM medium containing 10% FBS. Approximately 2×10⁴ cells were then seeded into a 24-well culture dish and cultured for 24 hours, and subjected to the lack of conditions for about 24 hours in DMEM medium with FBS removed.

在24小時之後，在用濃度為5 ng/ml之TGF-β處理之後，誘導細胞纖維化持續6小時。6小時後，細胞用製備實例2中所獲得之去鎂葉綠素酸a處理且進一步培養18小時(圖2)。接著，陰性對照組不用處理，陽性對照組僅用5 ng/ml濃度之TGF-β處理，且實驗組用5 ng/mL濃度之TGF-β處理，並在6小時後，用濃度為0.15 μM、0.3 μM、0.6 μM、1.2 μM、2.5 μM及5 μM之去鎂葉綠素酸a處理。After 24 hours, after treatment with TGF-β at a concentration of 5 ng/ml, cell fibrosis was induced for 6 hours. After 6 hours, the cells were treated with pheophytin a obtained in Preparation Example 2 and further cultured for 18 hours (Figure 2). Then, the negative control group was not treated, the positive control group was only treated with TGF-β at a concentration of 5 ng/ml, and the experimental group was treated with TGF-β at a concentration of 5 ng/mL, and after 6 hours, with a concentration of 0.15 μM , 0.3 μM, 0.6 μM, 1.2 μM, 2.5 μM and 5 μM pheophytin a treatment.

如下進行各樣品之免疫組織化學分析。首先，用PBS緩衝液洗滌樣品二次，且隨後添加4%甲醛以固定1小時，且使用抗膠原蛋白1A作為一級抗體且使用Alexa Fluor 488作為二級抗體。使用DAPI(4,6-二甲脒基-2-苯基吲哚)染色核，且使用螢光顯微鏡分析螢光強度之差異。The immunohistochemical analysis of each sample was performed as follows. First, the sample was washed twice with PBS buffer, and then 4% formaldehyde was added to fix for 1 hour, and anti-collagen 1A was used as the primary antibody and Alexa Fluor 488 was used as the secondary antibody. DAPI (4,6-dimethylamidino-2-phenylindole) was used to stain the nucleus, and a fluorescence microscope was used to analyze the difference in fluorescence intensity.

作為實驗之結果，如圖6中所示，藉由螢光強度證實膠原蛋白1A之表現在陰性對照CCD8-Lu細胞中較低且在用5 ng/ml TGF-β處理時顯著增加。另外，證實當用5 ng/ml TGF-β處理且6小時後用去鎂葉綠素酸a處理時，很大程度上抑制由TGF-β誘導之膠原蛋白1A之增加的表現；且特定言之，用2.5 μM或更高之濃度的去鎂葉綠素酸a處理展示對由TGF-β誘導之膠原蛋白1A之增加的表現的最佳抑制效果。因此，發現藉由去鎂葉綠素酸a抑制纖維化進展。實驗實例4.去鎂葉綠素酸a與肺纖維化治療劑之間抗纖維化活性的比較實驗實例4.1.使用定量即時聚合酶鏈反應比較抗纖維化活性As a result of the experiment, as shown in Figure 6, it was confirmed by fluorescence intensity that the expression of collagen 1A was lower in the negative control CCD8-Lu cells and significantly increased when treated with 5 ng/ml TGF-β. In addition, it was confirmed that when treated with 5 ng/ml TGF-β and treated with pheophytin a after 6 hours, the expression of the increase in collagen 1A induced by TGF-β was largely inhibited; and in particular, Treatment with pheophytin a at a concentration of 2.5 μM or higher showed the best inhibitory effect on the expression of the increase in collagen 1A induced by TGF-β. Therefore, it was found that the progression of fibrosis was inhibited by pheophytin a.Experimental Example 4. Comparison of anti-fibrotic activity between pheophytin a and a therapeutic agent for pulmonary fibrosisExperimental example 4.1. Comparison of anti-fibrotic activity using quantitative real-time polymerase chain reaction

使用定量即時聚合酶鏈反應進行基於細胞之分析實驗，以與先前批准作為肺纖維化治療劑之尼達尼布及吡非尼酮進行比較，分析製備實例2中獲得之去鎂葉綠素酸a的抗纖維化活性。Quantitative real-time polymerase chain reaction was used to perform cell-based analysis experiments to compare with the previously approved nintedanib and pirfenidone as therapeutic agents for pulmonary fibrosis. Anti-fibrotic activity.

在含有10% FBS之DMEM培養基中培養作為人類肺衍生纖維母細胞的CCD8-Lu細胞。隨後將約2×10⁵個細胞接種至6孔培養盤中，培養24小時，且在移除FBS之DMEM培養基中經受缺乏條件約24小時。CCD8-Lu cells, which are human lung-derived fibroblasts, were cultured in DMEM medium containing 10% FBS. Then, about 2×10⁵ cells were seeded into a 6-well culture dish, cultured for 24 hours, and subjected to the lack of conditions for about 24 hours in DMEM medium with FBS removed.

24小時後，在用濃度為5 ng/ml之TGF-β處理之後，誘導細胞纖維化持續6小時。6小時之後，細胞用製備實例2中所獲得之去鎂葉綠素酸a或肺纖維化治療劑處理且進一步培養18小時。接著，陰性對照組不用處理，陽性對照組僅用5 ng/ml濃度之TGF-β處理，且實驗組用濃度為5 ng/ml之TGF-β處理，並在6小時後，分別用濃度為2.5 μM或5 μM之去鎂葉綠素酸a、濃度為1 μM之尼達尼布及濃度為1 mM之吡非尼酮處理。After 24 hours, after treatment with TGF-β at a concentration of 5 ng/ml, cell fibrosis was induced for 6 hours. After 6 hours, the cells were treated with the pheophytin a or the therapeutic agent for pulmonary fibrosis obtained in Preparation Example 2 and further cultured for 18 hours. Then, the negative control group was not treated, the positive control group was only treated with TGF-β at a concentration of 5 ng/ml, and the experimental group was treated with TGF-β at a concentration of 5 ng/ml. After 6 hours, they were treated with TGF-β at a concentration of 5 ng/ml. Treatment with 2.5 μM or 5 μM pheophytin a, 1 μM nintedanib, and 1 mM pirfenidone.

在18小時之後，移除培養基，藉由添加PBS緩衝溶液將細胞洗滌二次，且直接向其中添加0.5 ml RNA提取物(trizol試劑，Thermo Fisher Scientific)，隨後使其在室溫下靜置10分鐘。隨後，添加0.1 ml氯仿且攪拌15秒，且隨後在約12,000×g下離心10分鐘。After 18 hours, the medium was removed, the cells were washed twice by adding a PBS buffer solution, and 0.5 ml of RNA extract (trizol reagent, Thermo Fisher Scientific) was directly added to it, and then allowed to stand atroom temperature 10 minute. Subsequently, 0.1 ml of chloroform was added and stirred for 15 seconds, and then centrifuged at about 12,000×g for 10 minutes.

分離上清液，添加相同體積異丙醇且以12,000×g離心10分鐘。隨後移除液體，且所得材料用75%乙醇洗滌一次，且隨後在室溫下乾燥。在乾燥之後，向其中添加約50 μl不含核糖核酸酶之純化蒸餾水，且使用分光光度計量測所得RNA之量及純度。The supernatant was separated, the same volume of isopropanol was added and centrifuged at 12,000×g for 10 minutes. The liquid was then removed, and the resulting material was washed once with 75% ethanol, and then dried at room temperature. After drying, add about 50 μl of purified distilled water without ribonuclease, and measure the amount and purity of the RNA obtained by spectrophotometry.

為使用所獲得之RNA合成cDNA，使2 μg經純化之總RNA在70℃下與寡聚dT進行退火反應5分鐘，且接著添加10×反轉錄緩衝溶液、10 mM dNTP、核糖核酸酶抑制劑及M-MLV反轉錄酶(Enzynomics，Korea)以在42℃下經60分鐘進行cDNA合成反應。In order to synthesize cDNA using the obtained RNA, 2 μg of purified total RNA was annealed with oligo dT at 70°C for 5 minutes, and then 10× reverse transcription buffer solution, 10 mM dNTP, ribonuclease inhibitor were added And M-MLV reverse transcriptase (Enzynomics, Korea) to perform a cDNA synthesis reaction at 42°C for 60 minutes.

在cDNA合成反應完成之後，逆轉錄酶藉由在72℃下加熱5分鐘失活，且隨後添加核糖核酸酶H以移除單股RNA且獲得最終cDNA。無論α-平滑肌肌動蛋白基因(其為肌纖維母細胞之特徵基因)、CCN2(或CTGF)基因(其為纖維化之特徵基因)、纖維連接蛋白(其為纖維化之特徵基因)及稱為反應性含氧物種之量之主要調節子的NOX4(NADPH氧化酶4)基因之表現是否改變，皆經由定量即時聚合酶鏈反應觀測到。一起定量GAPDH基因以校正表現差異。定量即時聚合酶鏈反應中所用之基因的核苷酸序列展示於下表2中。[表2]引子序列SEQ ID NOα-SMA-S5'-GCCCAGCCAAGCACTGTCAGGA-3'SEQ ID NO: 2α-SMA-AS5'-TCCCACCATCACCCCCTGATGTC-3'SEQ ID NO: 3CTGF-S5'-GGCTTACCGACTGGAAGAC-3'SEQ ID NO: 4CTGF-AS5'-AGGAGGCGTTGTCATTGG-3'SEQ ID NO: 5纖維連接蛋白-S5'-GTGGCTGAAGACACAAGGAA-3'SEQ ID NO: 6纖維連接蛋白-AS5'-CCTGCCATTGTAGGTGAAT-3'SEQ ID NO: 7NOX4-S5'-CACCTCTGCCTGTTCATCTG-3'SEQ ID NO: 8NOX4-AS5'-GGCTCTGCTTAGACACAATCC-3'SEQ ID NO: 9GAPDH-S5'-GTCTCCTCTGACTTCAACAGCG-3'SEQ ID NO: 10GAPDH-AS5'-ACCACCCTGTTGCTGTAGCCAA-3'SEQ ID NO: 11After the cDNA synthesis reaction was completed, the reverse transcriptase was inactivated by heating at 72°C for 5 minutes, and then ribonuclease H was added to remove single-stranded RNA and obtain the final cDNA. Regardless of α-smooth muscle actin gene (it is the characteristic gene of myofibroblasts), CCN2 (or CTGF) gene (it is the characteristic gene of fibrosis), fibronectin (it is the characteristic gene of fibrosis) and called Whether the expression of the NOX4 (NADPH oxidase 4) gene, which is the main regulator of the amount of reactive oxygen species, changes is observed through quantitative real-time polymerase chain reaction. Quantify GAPDH genes together to correct for differences in performance. The nucleotide sequences of the genes used in the quantitative real-time polymerase chain reaction are shown in Table 2 below. [Table 2]IntroductionsequenceSEQ ID NOα-SMA-S 5'-GCCCAGCCAAGCACTGTCAGGA-3' SEQ ID NO: 2α-SMA-AS 5'-TCCCACCATCACCCCCTGATGTC-3' SEQ ID NO: 3CTGF-S 5'-GGCTTACCGACTGGAAGAC-3' SEQ ID NO: 4CTGF-AS 5'-AGGAGGCGTTGTCATTGG-3' SEQ ID NO: 5Fibronectin-S 5'-GTGGCTGAAGACACAAGGAA-3' SEQ ID NO: 6Fibronectin-AS 5'-CCTGCCATTGTAGGTGAAT-3' SEQ ID NO: 7NOX4-S 5'-CACCTCTGCCTGTTCATCTG-3' SEQ ID NO: 8NOX4-AS 5'-GGCTCTGCTTAGACACAATCC-3' SEQ ID NO: 9GAPDH-S 5'-GTCTCCTCTGACTTCAACAGCG-3' SEQ ID NO: 10GAPDH-AS 5'-ACCACCCTGTTGCTGTAGCCAA-3' SEQ ID NO: 11

根據圖7至10中所示的實驗結果，發現當用TGF-β處理作為人類肺衍生纖維母細胞的CCD8-Lu細胞時，α-SMA、CTGF及NOX4基因之表現增加，且證實用濃度為2.5 μM及5 μM之去鎂葉綠素酸a處理顯著地抑制由TGF-β誘導之α-SMA、CTGF及NOX4基因之表現。另一方面，證實1 μM尼達尼布及1 mM吡非尼酮微弱地抑制或幾乎不抑制由TGF-β誘導之α-SMA、CTGF及NOX4基因之表現。因此，證實在相同條件下，相較於尼達尼布及吡非尼酮(用於肺纖維化之現有治療劑)，去鎂葉綠素酸a呈現出顯著極佳之抗纖維化活性。實驗實例4.2.基於纖維化相關蛋白質表現之抗纖維化活性之比較According to the experimental results shown in Figures 7 to 10, it was found that when CCD8-Lu cells, which are human lung-derived fibroblasts, were treated with TGF-β, the expression of α-SMA, CTGF, and NOX4 genes increased, and it was confirmed that the concentration used was 2.5 μM and 5 μM pheophytin a treatment significantly inhibited the expression of α-SMA, CTGF and NOX4 genes induced by TGF-β. On the other hand, it was confirmed that 1 μM nintedanib and 1 mM pirfenidone weakly or hardly inhibited the expression of α-SMA, CTGF and NOX4 genes induced by TGF-β. Therefore, it was confirmed that under the same conditions, compared with nintedanib and pirfenidone (existing therapeutic agents for pulmonary fibrosis), pheophytin a exhibited significantly and excellent anti-fibrotic activity.Experimental example 4.2. Comparison of anti-fibrosis activity based on fibrosis-related protein performance

使用西方墨點法分析進行基於細胞之分析實驗，以與先前經批准作為肺纖維化治療劑的尼達尼布及吡非尼酮相比較，分析去鎂葉綠素酸a之抗纖維化活性。Western blot analysis was used to perform cell-based analysis experiments to compare the anti-fibrotic activity of pheophytin a with nintedanib and pirfenidone, which were previously approved as therapeutic agents for pulmonary fibrosis.

在含有10% FBS之DMEM培養基中培養作為人類肺衍生纖維母細胞的CCD8-Lu細胞。隨後將約2×10⁵個細胞接種至6孔培養盤中，培養24小時，且在移除FBS之DMEM培養基中經受缺乏條件約24小時。CCD8-Lu cells, which are human lung-derived fibroblasts, were cultured in DMEM medium containing 10% FBS. Then, about 2×10⁵ cells were seeded into a 6-well culture dish, cultured for 24 hours, and subjected to the lack of conditions for about 24 hours in DMEM medium with FBS removed.

24小時後，在用濃度為5 ng/ml之TGF-β處理之後，誘導細胞纖維化持續6小時。隨後，細胞用製備實例2中所獲得之去鎂葉綠素酸a或肺纖維化治療劑處理且進一步培養18小時。接著，陰性對照組不用處理，陽性對照組僅用5 ng/ml濃度之TGF-β處理，且實驗組用濃度為5 ng/ml之TGF-β處理，並在6小時後，分別用濃度為2.5 μM或5 μM之去鎂葉綠素酸a、濃度為1 μM之尼達尼布及濃度為1 mM之吡非尼酮處理。After 24 hours, after treatment with TGF-β at a concentration of 5 ng/ml, cell fibrosis was induced for 6 hours. Subsequently, the cells were treated with the pheophytin a or the therapeutic agent for pulmonary fibrosis obtained in Preparation Example 2 and further cultured for 18 hours. Then, the negative control group was not treated, the positive control group was only treated with TGF-β at a concentration of 5 ng/ml, and the experimental group was treated with TGF-β at a concentration of 5 ng/ml. After 6 hours, they were treated with TGF-β at a concentration of 5 ng/ml. Treatment with 2.5 μM or 5 μM pheophytin a, 1 μM nintedanib, and 1 mM pirfenidone.

18小時後，移除培養基且藉由添加PBS緩衝溶液洗滌細胞二次，且直接向其中添加含有蛋白酶抑制劑之100 μl RIPA緩衝溶液(150 mM NaCl、1% NP-40、0.5%去氧膽酸、0.1% SDS、50 mM Tris pH 7.5)。10分鐘後，使用刮刀回收細胞且轉移至微管中且以約12,000×g離心10分鐘。After 18 hours, remove the medium and wash the cells twice by adding PBS buffer solution, and directly add 100 μl RIPA buffer solution containing protease inhibitors (150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid) to it. Acid, 0.1% SDS, 50 mM Tris pH 7.5). After 10 minutes, the cells were recovered using a spatula and transferred to a microtube and centrifuged at approximately 12,000×g for 10 minutes.

獲得經分離之上清液且轉移至新的微管中，且接著使用二喹啉甲酸分析(bicinchoninic acid assay，BCA分析)蛋白質定量套組來定量該蛋白質。對具有相同量蛋白質之各樣品進行電泳，且隨後進行西方墨點法。使用抗α-SMA、抗膠原蛋白1A及抗纖維連接蛋白作為一級抗體，且使用抗小鼠IgG HRP或抗兔IgG HRP作為二級抗體。使用抗β-肌動蛋白抗體校正蛋白質表現之差異。The separated supernatant was obtained and transferred to a new microtube, and then the protein was quantified using the bicinchoninic acid assay (BCA analysis) protein quantification kit. Electrophoresis was performed on each sample with the same amount of protein, and then the Western blot method was performed. Anti-α-SMA, anti-collagen 1A, and anti-fibronectin were used as primary antibodies, and anti-mouse IgG HRP or anti-rabbit IgG HRP were used as secondary antibodies. Use anti-β-actin antibodies to correct for differences in protein performance.

根據圖11中所示的實驗結果，發現當用TGF-β處理作為人類肺衍生纖維母細胞的CCD8-Lu細胞時，α-SMA、膠原蛋白1A及纖維連接蛋白之表現增加，且證實用濃度為2.5 μM及5 μM之去鎂葉綠素酸a處理顯著地抑制由TGF-β誘導之α-SMA、膠原蛋白1A及纖維連接蛋白之表現。另一方面，證實1 μM尼達尼布及1 mM吡非尼酮微弱地抑制或幾乎不抑制由TGF-β誘導之α-SMA、膠原蛋白1A及纖維連接蛋白之表現。因此，證實在相同條件下，相較於尼達尼布及吡非尼酮(用於肺纖維化之現有治療劑)，去鎂葉綠素酸a呈現出顯著極佳之抗纖維化活性。實驗實例4.3.使用免疫細胞化學染色分析進行對抗纖維化活性之分析According to the experimental results shown in Figure 11, it was found that when CCD8-Lu cells, which are human lung-derived fibroblasts, were treated with TGF-β, the expression of α-SMA, collagen 1A, and fibronectin increased, and it was confirmed that the concentration was used The 2.5 μM and 5 μM pheophytin a treatment significantly inhibited the expression of α-SMA, collagen 1A and fibronectin induced by TGF-β. On the other hand, it was confirmed that 1 μM nintedanib and 1 mM pirfenidone weakly or hardly inhibited the expression of α-SMA, collagen 1A and fibronectin induced by TGF-β. Therefore, it was confirmed that under the same conditions, compared with nintedanib and pirfenidone (existing therapeutic agents for pulmonary fibrosis), pheophytin a exhibited significantly and excellent anti-fibrotic activity.Experimental example 4.3. Analysis of anti-fibrosis activity using immunocytochemical staining analysis

使用免疫細胞化學染色方法進行基於細胞之分析實驗，以與先前經批准作為肺纖維化治療劑的尼達尼布及吡非尼酮相比較，分析去鎂葉綠素酸a之抗纖維化活性。The immunocytochemical staining method was used to conduct cell-based analysis experiments to compare with previously approved nintedanib and pirfenidone as therapeutic agents for pulmonary fibrosis to analyze the anti-fibrotic activity of pheophytin a.

在含有10% FBS之DMEM培養基中培養作為人類肺衍生纖維母細胞的CCD8-Lu細胞。隨後將約2×10⁴個細胞接種至24孔培養盤中且培養24小時，且在移除FBS之DMEM培養基中經受缺乏條件約24小時。CCD8-Lu cells, which are human lung-derived fibroblasts, were cultured in DMEM medium containing 10% FBS. Approximately 2×10⁴ cells were then seeded into a 24-well culture dish and cultured for 24 hours, and subjected to the lack of conditions for about 24 hours in DMEM medium with FBS removed.

在24小時之後，在用濃度為5 ng/ml之TGF-β處理之後，誘導細胞纖維化持續6小時。在6小時之後，細胞用2.5 μM或5 μM去鎂葉綠素酸a、1 μM尼達尼布或1 mM吡非尼酮處理。After 24 hours, after treatment with TGF-β at a concentration of 5 ng/ml, cell fibrosis was induced for 6 hours. After 6 hours, the cells were treated with 2.5 μM or 5 μM pheophytin a, 1 μM nintedanib, or 1 mM pirfenidone.

如下進行各樣品之免疫組織化學分析。首先，用PBS緩衝液洗滌樣品二次，且隨後添加4%甲醛以固定1小時，且使用抗膠原蛋白1A作為一級抗體且使用Alexa Fluor 488作為二級抗體。使用DAPI(4,6-二甲脒基-2-苯基吲哚)染色核，且使用螢光顯微鏡分析螢光強度之差異。The immunohistochemical analysis of each sample was performed as follows. First, the sample was washed twice with PBS buffer, and then 4% formaldehyde was added to fix for 1 hour, and anti-collagen 1A was used as the primary antibody and Alexa Fluor 488 was used as the secondary antibody. DAPI (4,6-dimethylamidino-2-phenylindole) was used to stain the nucleus, and a fluorescence microscope was used to analyze the difference in fluorescence intensity.

作為實驗之結果，如圖12中所示，藉由螢光強度證實膠原蛋白1A之表現在陰性對照CCD8-Lu細胞中較低且在用5 ng/ml TGF-β處理時顯著增加。另外，證實當用5 ng/ml TGF-β處理並在6小時後用去鎂葉綠素酸a處理時，由TGF-β誘導之膠原蛋白1A之增加的表現在去鎂葉綠素酸a濃度為2.5 μM及5 μM時得到顯著抑制。另一方面，證實用1 μM尼達尼布處理微弱地抑制由TGF-β誘導之膠原蛋白1A蛋白質之表現。因此，證實在相同條件下，與尼達尼布及吡非尼酮(用於肺纖維化之現有治療劑)相比，去鎂葉綠素酸a呈現出顯著極佳之抗纖維化活性。實驗實例5.證實與纖維化相關之磷酸化蛋白質之表現調節As a result of the experiment, as shown in Figure 12, it was confirmed by fluorescence intensity that the expression of collagen 1A was lower in the negative control CCD8-Lu cells and significantly increased when treated with 5 ng/ml TGF-β. In addition, it was confirmed that when treated with 5 ng/ml TGF-β and treated with pheophytin a after 6 hours, the increase in collagen 1A induced by TGF-β was manifested at a concentration of pheophytin a of 2.5 μM And it was significantly inhibited at 5 μM. On the other hand, it was confirmed that treatment with 1 μM nintedanib weakly inhibited the expression of collagen 1A protein induced by TGF-β. Therefore, it was confirmed that under the same conditions, compared with nintedanib and pirfenidone (existing therapeutic agents for pulmonary fibrosis), pheophytin a exhibited significantly and excellent anti-fibrotic activity.Experimental Example 5. Confirmation of the expression regulation of phosphorylated proteins related to fibrosis

使用西方墨點法分析進行基於細胞之分析實驗，以確定去鎂葉綠素酸a對TGF-β信號傳導路徑中之磷酸化的影響。Western blot analysis was used to perform cell-based analysis experiments to determine the effect of pheophytin a on phosphorylation in the TGF-β signaling pathway.

使用西方墨點法分析進行基於細胞之分析實驗，以與先前經批准作為肺纖維化治療劑的尼達尼布及吡非尼酮相比較，分析去鎂葉綠素酸a之抗纖維化活性。Western blot analysis was used to perform cell-based analysis experiments to compare the anti-fibrotic activity of pheophytin a with nintedanib and pirfenidone, which were previously approved as therapeutic agents for pulmonary fibrosis.

在含有10% FBS之DMEM培養基中培養作為人類肺衍生纖維母細胞的CCD8-Lu細胞。隨後將約2×10⁵個細胞接種至6孔培養盤中，培養24小時，且在移除FBS之DMEM培養基中經受缺乏條件約24小時。CCD8-Lu cells, which are human lung-derived fibroblasts, were cultured in DMEM medium containing 10% FBS. Then, about 2×10⁵ cells were seeded into a 6-well culture dish, cultured for 24 hours, and subjected to the lack of conditions for about 24 hours in DMEM medium with FBS removed.

24小時後，在用濃度為5 ng/ml之TGF-β處理之後，誘導細胞纖維化持續6小時。隨後，細胞用製備實例2中所獲得之去鎂葉綠素酸a或肺纖維化治療劑處理且進一步培養18小時。接著，陰性對照組不用處理，陽性對照組僅用5 ng/ml濃度之TGF-β處理，且實驗組用5 ng/ml濃度之TGF-β處理，並在6小時後分別用5 μM濃度之去鎂葉綠素酸a及1 μM濃度之尼達尼布處理。After 24 hours, after treatment with TGF-β at a concentration of 5 ng/ml, cell fibrosis was induced for 6 hours. Subsequently, the cells were treated with the pheophytin a or the therapeutic agent for pulmonary fibrosis obtained in Preparation Example 2 and further cultured for 18 hours. Then, the negative control group was not treated, the positive control group was only treated with TGF-β at a concentration of 5 ng/ml, and the experimental group was treated with TGF-β at a concentration of 5 ng/ml. After 6 hours, they were treated with TGF-β at a concentration of 5 μM. Treatment with pheophytin-a and nintedanib at a concentration of 1 μM.

1小時後，移除培養基且藉由添加PBS緩衝溶液洗滌細胞二次，且直接向其中添加含有蛋白酶抑制劑之100 μl RIPA緩衝溶液(150 mM NaCl、1% NP-40、0.5%去氧膽酸、0.1% SDS、50 mM Tris pH 7.5)。10分鐘後，使用刮刀回收細胞且轉移至微管中且以約12,000×g離心10分鐘。After 1 hour, remove the medium and wash the cells twice by adding PBS buffer solution, and directly add 100 μl RIPA buffer solution containing protease inhibitors (150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid) to it. Acid, 0.1% SDS, 50 mM Tris pH 7.5). After 10 minutes, the cells were recovered using a spatula and transferred to a microtube and centrifuged at approximately 12,000×g for 10 minutes.

獲得經分離之上清液且轉移至新的微管中，且接著使用二喹啉甲酸分析(bicinchoninic acid assay，BCA分析)蛋白質定量套組來定量該蛋白質。對具有相同量蛋白質之各樣品進行電泳，且隨後進行西方墨點法。使用抗pSmad3抗體、抗Smad3抗體、抗pERK抗體及抗ERK抗體作為一級抗體，且使用抗小鼠IgG HRP或抗兔IgG HRP作為二級抗體。The separated supernatant was obtained and transferred to a new microtube, and then the protein was quantified using the bicinchoninic acid assay (BCA analysis) protein quantification kit. Electrophoresis was performed on each sample with the same amount of protein, and then the Western blot method was performed. Anti-pSmad3 antibody, anti-Smad3 antibody, anti-pERK antibody, and anti-ERK antibody were used as primary antibodies, and anti-mouse IgG HRP or anti-rabbit IgG HRP were used as secondary antibodies.

根據圖13中所示之實驗結果，發現當用TGF-β處理作為人類肺衍生纖維母細胞的CCD8-Lu細胞時磷酸化Smad3及磷酸化ERK之表現增加，且證實用濃度為5 μM之去鎂葉綠素酸a處理顯著地抑制由TGF-β誘導之磷酸化Smad3及磷酸化ERK的表現。另一方面，證實1 μM尼達尼布幾乎不抑制由TGF-β誘導之磷酸化Smad3及磷酸化ERK之表現。因此，證實去鎂葉綠素酸a藉由抑制由TGF-β誘導之磷酸化Smad3及磷酸化ERK蛋白之表現而呈現出抗纖維化功效。實驗實例6.鑑別肺纖維化模型小鼠中之去鎂葉綠素酸a的抗纖維化效果According to the experimental results shown in Figure 13, it was found that when CCD8-Lu cells, which are human lung-derived fibroblasts, were treated with TGF-β, the expression of phosphorylated Smad3 and phosphorylated ERK increased, and it was confirmed that a concentration of 5 μM was used. Pheophyllin a treatment significantly inhibited the expression of phosphorylated Smad3 and phosphorylated ERK induced by TGF-β. On the other hand, it was confirmed that 1 μM nintedanib hardly inhibited the expression of phosphorylated Smad3 and phosphorylated ERK induced by TGF-β. Therefore, it was confirmed that pheophytin-a exhibits anti-fibrotic effects by inhibiting the expression of phosphorylated Smad3 and phosphorylated ERK protein induced by TGF-β.Experimental Example 6. Identification of the anti-fibrotic effect of pheophytin a in pulmonary fibrosis model mice

在此實驗中，評估肺纖維化模型中去鎂葉綠素酸a之抗纖維化效果，其中肺纖維化係藉由用博萊黴素(bleomycin，BLM)(NIPPON KAYAKU, KIT編碼號NA)處理C57BL/6NCrl0ri小鼠(Orient Bio)誘導。如下表3中所示製備實驗組。[表3]組性別動物數量滴注體積(μl)BLM劑量(mg/kg)經口投予體積(ml/kg)有效物質(mg/kg)尼達尼布劑量(mg/kg)媒劑對照組雄性550-10--陰性對照組(纖維化對照組)雄性5501.810--T1雄性5501.8101.25 mpk-T2雄性5501.8105 mpk-T3雄性5501.81020 mpk-T4雄性5501.81080 mpk-陽性對照組(尼達尼布)雄性5501.81060 mpk60In this experiment, the anti-fibrotic effect of pheophytin a in a pulmonary fibrosis model was evaluated, in which pulmonary fibrosis was treated with bleomycin (BLM) (NIPPON KAYAKU, KIT code number NA) to treat C57BL /6NCrl0ri mice (Orient Bio) induced. The experimental groups were prepared as shown in Table 3 below. [table 3]GroupgenderNumber of animalsInstillation volume(μl)BLMdose(mg/kg)Oral administration volume(ml/kg)Effective substance(mg/kg)Nintedanib dose(mg/kg)Vehiclecontrol group male 5 50 - 10 - -Negative control group(fibrosis control group) male 5 50 1.8 10 - -T1 male 5 50 1.8 10 1.25 mpk -T2 male 5 50 1.8 10 5 mpk -T3 male 5 50 1.8 10 20 mpk -T4 male 5 50 1.8 10 80 mpk -Positive control group(nintedanib) male 5 50 1.8 10 60mpk 60

對小鼠進行稱重且分成總共七組，且接著在第1天，在媒劑對照組中用無菌生理鹽水及在除媒劑對照組外之其他組中用BLM經氣管內投予以誘導肺纖維化。接著，自第1天至第21天，向媒劑對照組及陰性對照組(纖維化對照組)投予不含有效物質的DMSO(σ D2650-5X)。The mice were weighed and divided into a total of seven groups, and then onday 1, the lungs were induced by intratracheal administration of BLM in the vehicle control group with sterile saline and in the other groups except the vehicle control group Fibrosis. Then, fromday 1 today 21, the vehicle control group and the negative control group (fibrosis control group) were administered DMSO (σ D2650-5X) without an effective substance.

另外，T1至T4組及陽性對照組分別每天一次經口投予有效物質(去鎂葉綠素酸a)及尼達尼布。然而，在第1天，在經氣管內投予BLM一小時之前經口投予有效物質或賦形劑。In addition, the T1 to T4 groups and the positive control group were orally administered the effective substances (depheophyllin a) and nintedanib once a day, respectively. However, onday 1, the effective substance or excipient was orally administered one hour before the intratracheal administration of BLM.

在投予之後，每天一次觀測死亡及瀕死狀態，且每天二次觀測諸如外觀及行為變化之一般症狀。當動物被判定為異常時，則以較寬之範圍、增加之頻率對其進行觀測，且當展示與嚴重病痛或死亡相當之毒性時，使其安樂死。在投予期間及在屍檢日總計進行7次(第1天、第2天、第4天、第8天、第11天、第15天及第18天)體重量測。After the administration, death and near-death status were observed once a day, and general symptoms such as changes in appearance and behavior were observed twice a day. When an animal is judged to be abnormal, it is observed with a wider range and increased frequency, and when it exhibits toxicity equivalent to severe illness or death, it is euthanized. A total of 7 body weight measurements (day 1,day 2, day 4, day 8, day 11,day 15 and day 18) were performed during the administration period and on the day of autopsy.

在第22天，動物藉由吸入異氟醚而安樂死且進行屍檢。首先，切割後大靜脈及隱靜脈以釋放血液以放血，觀測到胸腔及腹腔外觀存在異常，且接著取出肺。對所取之肺進行稱重且用10%中性緩衝福馬林溶液固定其一部分(左肺)以用於組織病理學檢查。組織樣本由所固定之肺製備，用蘇木精及伊紅(hematoxylin&eosin，H&E)染色，且經受組織病理學檢查。所取之肺的器官重量比藉由使用以下計算式計算。＜等式1＞器官重量比=總肺重量(g)/總體重(g)Onday 22, the animals were euthanized by isoflurane inhalation and an autopsy was performed. First, the large veins and saphenous veins were cut to release blood for exsanguination, abnormal appearance of the thoracic cavity and abdominal cavity was observed, and then the lungs were taken out. The taken lung was weighed and a part of it (left lung) was fixed with 10% neutral buffered formalin solution for histopathological examination. Tissue samples were prepared from the fixed lung, stained with hematoxylin and eosin (H&E), and subjected to histopathological examination. The lung organ weight ratio is calculated by using the following calculation formula.<Equation 1>Organ weight ratio = total lung weight (g)/total weight (g)

實驗之結果由平均值及標準差表示且使用Pristima系統或統計分析系統(SAS/STAT)以統計方式分析。The results of the experiment are represented by the average value and standard deviation and analyzed statistically using the Pristima system or statistical analysis system (SAS/STAT).

在使用Pristima系統進行統計分析之情況下，藉由多重比較分析進行各組之間的比較。實驗資料使用巴特勒特測試(Bartlett's Test)進行變異數檢定之均質性，且接著藉由單因子變異數分析(One-Way Analysis of Variance，ANOVA)測試等變異性資料且藉由鄧尼特測試(Dunnett's Test)分析組間差異。非等變異性資料藉由克拉斯卡-瓦立斯測試(Kruskal-Wallis Test)來分析，且投予組與對照組之間的差異藉由鄧氏秩和檢定(Dunn's Rank Sum Test)來分析。或者，對於二組之間的變異數檢定之均質性進行F-測試。In the case of statistical analysis using the Pristima system, comparisons between groups are performed by multiple comparison analysis. The experimental data uses the Bartlett's Test to perform the homogeneity of the variance test, and then the variability data is tested by One-Way Analysis of Variance (ANOVA) and is tested by the Dunnett test (Dunnett's Test) Analyze differences between groups. The non-equal variability data was analyzed by the Kruskal-Wallis Test, and the difference between the administration group and the control group was analyzed by Dunn's Rank Sum Test . Alternatively, perform an F-test for the homogeneity of the variance test between the two groups.

在使用SAS/STAT之統計分析中，二組之間的變異數檢定之均質性使用F測試進行。等變異性資料經受史都登氏t測試(Student's t-test)以驗證組間差異，且非等變異性資料經受威爾卡遜秩和檢定(Wilcoxon Rank Sum Test)。實驗結果之統計分析方法概述於表4中。[表4]所測試之參數統計分析之方法所測試之參數初步測試不顯著顯著體重巴特勒特測試F測試 ANOVA測試，鄧尼特測試史都登氏t測試克拉斯卡-瓦立斯測試，鄧氏秩和檢定威爾卡遜秩和檢定體重增加器官重量In the statistical analysis using SAS/STAT, the homogeneity of the variance test between the two groups was performed using the F test. The data of equal variability were subjected to Student's t-test to verify the differences between groups, and the data of non-equal variability were subjected to the Wilcoxon Rank Sum Test (Wilcoxon Rank Sum Test). The statistical analysis methods of the experimental results are summarized in Table 4. [Table 4]Parameters testedMethods of statistical analysisParameters tested Preliminary test Not significant Significantweight Butler test F test ANOVA test, Dunnett test, Studden's t test Krasca-Wallis test, Dunn’s rank sum test, Wilkason rank sum testWeight gainOrgan weight

因此，證實在肺纖維化模型中，投予去鎂葉綠素酸a之組展示出相比於陰性對照組之抗纖維化效果。另外，證實即使與投予尼達尼布之陽性對照組相比，投予去鎂葉綠素酸a之組亦展示顯著抗纖維化效果。Therefore, it was confirmed that in the pulmonary fibrosis model, the group administered with pheophytin a showed an anti-fibrotic effect compared to the negative control group. In addition, it was confirmed that even when compared with the positive control group administered with nintedanib, the group administered with pheophytin a also exhibited a significant anti-fibrotic effect.

(無)(without)

圖1展示自黃漆木(Dendropanax Morbiferus)分離去鎂葉綠素酸a之方法。Figure 1 shows the method of separating chlorophyllin a from yellow lacquered wood (Dendropanax Morbiferus).

圖2為用於分析在細胞層面之去鎂葉綠素酸a之抗纖維化活性的實驗之示意圖。Figure 2 is a schematic diagram of an experiment used to analyze the anti-fibrotic activity of pheophytin a at the cell level.

圖3為作為人類肺纖維母細胞之CCD8-Lu及LL-29中的去鎂葉綠素酸a之細胞毒性的實驗結果。Figure 3 shows the cytotoxicity test results of pheophytin a in CCD8-Lu and LL-29 as human lung fibroblasts.

圖4為用於測定根據用TGF-β及/或去鎂葉綠素酸a處理之Smad蛋白之DNA結合能力差異的螢光素酶分析之結果。Figure 4 is the result of luciferase analysis for determining the difference in DNA binding ability of Smad proteins treated with TGF-β and/or pheophytin a.

圖5為藉由使用西方墨點法，用不同濃度TGF-β及/或去鎂葉綠素酸a處理CCD8-Lu細胞(其為人類肺纖維母細胞)來比較膠原蛋白1A、纖維連接蛋白及α-平滑肌肌動蛋白蛋白之表現位準差異的結果。Figure 5 shows the comparison of collagen 1A, fibronectin and α by treating CCD8-Lu cells (which are human lung fibroblasts) with different concentrations of TGF-β and/or pheophytin a by using the Western ink dot method. -The result of the difference in the expression level of smooth muscle actin protein.

圖6為藉由使用免疫細胞化學分析，用不同濃度之TGF-β及/或去鎂葉綠素酸a處理CCD8-Lu細胞(其為人類肺纖維母細胞)來比較膠原蛋白1A之表現位準差異的結果。Figure 6 compares the level of expression of collagen 1A by treating CCD8-Lu cells (which are human lung fibroblasts) with different concentrations of TGF-β and/or pheophytin a by immunocytochemical analysis the result of.

圖7至圖10為藉由使用定量聚合酶鏈反應(qRT-PCR)，用TGF-β及去鎂葉綠素酸a、尼達尼布或吡非尼酮處理CCD8-Lu細胞(其為人類肺纖維母細胞)來比較α-SMA基因(圖7)、CTGF基因(圖8)、纖維連接蛋白基因(圖9)及NOX4基因(圖10)之表現位準差異的結果。Figures 7 to 10 show the treatment of CCD8-Lu cells (which are human lungs) with TGF-β and pheophytin a, nintedanib or pirfenidone by using quantitative polymerase chain reaction (qRT-PCR) Fibroblasts) to compare the results of the expression level differences of α-SMA gene (Figure 7), CTGF gene (Figure 8), fibronectin gene (Figure 9) and NOX4 gene (Figure 10).

圖11為藉由使用西方墨點法，用TGF-β及去鎂葉綠素酸a、尼達尼布或吡非尼酮處理CCD8-Lu細胞(其為人類肺纖維母細胞)來比較膠原蛋白1A、纖維連接蛋白及α-平滑肌肌動蛋白蛋白之表現位準差異的結果。Figure 11 shows the comparison of collagen 1A by treating CCD8-Lu cells (which are human lung fibroblasts) with TGF-β and pheophytin-a, nintedanib or pirfenidone using the Western blot method , Fibronectin and α-Smooth muscle actin protein expression level difference results.

圖12為藉由使用免疫細胞化學分析，用TGF-β及去鎂葉綠素酸a或尼達尼布處理CCD8-Lu細胞(其為人類肺纖維母細胞)來比較膠原蛋白1A蛋白之表現位準差異的結果。Figure 12 shows the comparison of the expression level of collagen 1A protein by treating CCD8-Lu cells (which are human lung fibroblasts) with TGF-β and pheophytin a or nintedanib by using immunocytochemical analysis The result of the difference.

圖13為藉由使用西方墨點法，用TGF-β及去鎂葉綠素酸a或尼達尼布處理CCD8-Lu細胞(其為人類肺纖維母細胞)來比較Smad、磷酸化Smad3、ERK及磷酸化ERK蛋白之表現位準差異的結果。Figure 13 shows the comparison of Smad, phosphorylated Smad3, ERK and CCD8-Lu cells (which are human lung fibroblasts) treated with TGF-β and pheophytin a or nintedanib by using the Western blot method. The result of the difference in the level of expression of phosphorylated ERK protein.

Claims (8)

Translated fromChinese

一種用於預防或治療纖維化之藥學組成物，其含有作為一活性成分之一去鎂葉綠素酸化合物。A pharmaceutical composition for preventing or treating fibrosis, which contains a pheophytin acid compound as one of the active ingredients.如請求項1之藥學組成物，其中該去鎂葉綠素酸化合物為去鎂葉綠素酸a。The pharmaceutical composition of claim 1, wherein the pheophytin acid compound is pheophytin a.如請求項1之藥學組成物，其中該去鎂葉綠素酸化合物抑制由TGF-β誘導之Smad及ERK的磷酸化。The pharmaceutical composition of claim 1, wherein the pheophytin acid compound inhibits the phosphorylation of Smad and ERK induced by TGF-β.如請求項1之藥學組成物，其中該去鎂葉綠素酸化合物抑制由TGF-β誘導之α-平滑肌肌動蛋白、纖維連接蛋白或膠原蛋白之表現。The pharmaceutical composition of claim 1, wherein the pheophytin acid compound inhibits the expression of α-smooth muscle actin, fibronectin or collagen induced by TGF-β.如請求項1之藥學組成物，其中該纖維化為選自由以下組成之群組的任意一或多者：肝纖維化、肺纖維化、皮膚纖維化、關節纖維化、神經纖維化、胰纖維化、肌肉纖維化及腹膜纖維化。The pharmaceutical composition of claim 1, wherein the fibrosis is any one or more selected from the group consisting of liver fibrosis, pulmonary fibrosis, skin fibrosis, joint fibrosis, neurofibrosis, pancreatic fiber Fibrosis, muscle fibrosis and peritoneal fibrosis.一種用於預防或改善纖維化之食品組成物，其含有作為一活性成分之一去鎂葉綠素酸化合物。A food composition for preventing or improving fibrosis, which contains a pheophytin acid compound as one of the active ingredients.如請求項6之食品組成物，其中該去鎂葉綠素酸化合物為去鎂葉綠素酸a。The food composition of claim 6, wherein the pheophytin acid compound is pheochlorophyll acid a.一種預防及治療在一個體之纖維化的方法，其包含向該個體投予一去鎂葉綠素酸化合物。A method for preventing and treating fibrosis in an individual, which comprises administering a pheophytin acid compound to the individual.

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